Enzymatic and Antimicrobial Activity of Biologically Active Samples from Aloe arborescens and Aloe barbadensis

Simple Summary Antimicrobial resistance is one of the major threats to public health, and additional concerns are reduced efficacy and increased toxicity of synthetically derived drugs. Hence, it is all the more important to research new antimicrobials derived from natural sources. Aloe spp. have long been acknowledged in traditional medicine, as their ability of treating skin and digestive problems, wound healing, anti-inflammatory, antimicrobial and other promising properties are known. This study presents the content of various bioactive substances in samples of two Aloe spp., Aloe arborescens and Aloe barbadensis, and their enzymatic, antioxidant and antimicrobial activity. Obtained bioactive compounds with antimicrobial effect have a huge potential to inhibit the growth of microorganisms that are extremely susceptible to gaining resistance and could be used in versatile applications in the cosmetics, food, medical and pharmaceutical industries. Abstract Recently, the use of Aloe species has become very widespread. These are extensively used as a nutraceutical in a variety of health care products and food supplements. In addition, the occurrence of the quickly adaptable microorganisms, particularly bacteria, which can develop resistance to antibiotics, is a major problem for public health, and therefore, it is necessary to search for new antimicrobials. In our study, the content of total phenols, proanthocyanidins, and proteins in fresh and lyophilized samples of A. arborescens and A. barbadensis and their ethanol extracts was investigated. Furthermore, enzymatic and antioxidant activity of samples were studied. Since antimicrobial activity of fresh samples was determined in our latest research, a more detailed study of antimicrobial effectiveness of A. arborescens and A. barbadensis (lyophilized, extracts) was performed. Ethanol extracts in particular contain higher concentrations of bioactive substances and show the topmost antioxidant activity. The novelty of the study refers to the observation of industrially important enzyme activities such as α-amylase, cellulase, lipase, peroxidase, protease, and transglutaminase in the samples as well as the microbial growth inhibition rates determination (MGIR) at different concentrations of added aloe samples. All samples inhibited the growth of all tested microbial cells. MIC90 for A. arborescens and A. barbadensis were also determined in case of B. cereus, P. aeruginosa, P. fluorescens, and S. aureus. The results of our study tend to give credence to the popular use of both aloes in medicine and in the cosmetic, food, and pharmaceutical industries.


Introduction
Much of today's biomass on planet Earth is represented by microbes. Although they are not perceived by the human eye, they are extremely influential in people's lives. Among other things, many microbes constitute the natural microbial flora in the human body. Microbes are important for the functioning of the human body because they participate in the food chain and other important processes [1,2]. The problem are pathogenic microorganisms. These are dangerous to humans and can cause diseases. Diseases caused by pathogens are a major problem for human health. Antibiotics that treat bacterial infections are no longer so effective because of microorganisms' property of being able to develop drug resistance. Especially bacteria adapt quickly and therefore negate the effects of drugs [3][4][5].
Because drugs can have a toxic effect on humans, there is a growing interest in alternative, natural antimicrobial agents that would inhibit the growth and reproduction of opportunistic bacteria. Plants and natural preparations that have potential to inhibit the growth of microorganisms and ensure lower toxicity than drugs are nowadays very interesting [6]. Among these plants are also Aloe arborescens and Aloe barbadensis. A. barbadensis is well known as Aloe vera [7], while generally A. arborescens is less known. Although most people are unfamiliar with it, several countries have been using A. arborescens for many years in traditional medicine [8]. Both have many beneficial health effects ( Figure 1) and can be used for different purposes [9]. Sánchez et al. [10] recently published a review on the updated pharmacological properties of A. barbadensis and its main active ingredients.

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barbadensis is well known as Aloe vera [7], while generally A. arborescens is less known. Although most people are unfamiliar with it, several countries have been using A. arborescens for many years in traditional medicine [8]. Both have many beneficial health effects ( Figure 1) and can be used for different purposes [9]. Sánchez et al. [10] recently published a review on the updated pharmacological properties of A. barbadensis and its main active ingredients. Most commonly, both aloes are used to treat skin problems, abdominal problems, digestive problems, for the healing of wounds and burns, and as an anti-inflammatory and antimicrobial agent [16,17]. Because aloes were thus used in traditional medicine, the food industry took advantage of this and began using aloe extracts in food and dietary supplements as a functional food [18,19]. Lately, the aloe-derived gel has become increasingly popular. In most cases, it is used in the cosmetics industry and the pharmaceutical industry and as a nutraceutical in the food industry [16,20,21].
A. barbadensis contains mostly water and approx. 0.7% is represented by various bioactive substances, of which 75 are known [22,23]. These include nutritive constituents as carbohydrates (polysaccharides called glucomannans, etc.), vitamins (ascorbic acid, carotenoids, tocopherols, vitamin B1, B2, B6, niacin, folic acid, etc.), enzymes (cellulase, carboxypeptidase, amylase, bradykinase, oxidase, and catalase, etc.), minerals (magnesium, calcium, iron, copper, zinc, and chromium, etc.), proteins (glycoproteins), and amino acids (seven essential amino acids and additionally 20 out of 22 amino acids that occur naturally). Nonnutritive constituents include phenolic compounds (e.g., anthraquinones), organic acids (salicylic, lactic, acetic, malic, and succinic acid, etc.), and phytosterols (lupeol, cholesterol, β-sitosterol, campesterol, etc.) [24][25][26]. Certain molecules in aloe have antibacterial, antiviral, and antifungal properties. Above all, A. vera is able to slow down the development of bacteria and fungi [27] due to the presence of two organic acids, cinnamic and chrysophanic acid also known as chrysophanol [28]. The anthraquinone group also Most commonly, both aloes are used to treat skin problems, abdominal problems, digestive problems, for the healing of wounds and burns, and as an anti-inflammatory and antimicrobial agent [16,17]. Because aloes were thus used in traditional medicine, the food industry took advantage of this and began using aloe extracts in food and dietary supplements as a functional food [18,19]. Lately, the aloe-derived gel has become increasingly popular. In most cases, it is used in the cosmetics industry and the pharmaceutical industry and as a nutraceutical in the food industry [16,20,21].
"Medicinal aloe" is another name for A. arborescens as it has been used as a folk medicine in many countries around the world for many years [42]. A. arborescens has great potential, but not much research in this field has been done. Our published article [1] based on the study and comparison of the inhibitory effect of natural gel and juice of A. arborescens and A. barbadensis brings a very important contribution to this field. As the exceptional antimicrobial efficacy of all-natural gels and juices from both aloes was found, we continued with research in this area. Therefore, the focus of our presented research was to study the content of various bioactive secondary metabolites that could also contribute to the inhibitory effect of these aloe species and to further study antimicrobial activity of A. arborescens and A. barbadensis on some opportunistic bacteria and fungi. Tested microorganisms are important human and animal pathogens and often represent groups of major infectious agents, i.e., Gram-positive bacteria (B. cereus, S. aureus), Gram-negative bacteria (E. coli, P. aeruginosa, and P. fluorescens), and yeasts (C. albicans). For all of these pathogens, a large increase in resistance to commonly used drugs was observed [43,44]. Many antimicrobial studies have been done in the last few years, mainly with A. barbadensis. Recently, Forno-Bell et al. [45] showed antibacterial activity of methanolic extract of A. barbadensis on S. aureus, E. coli, Streptococcus uberis, and MRSA cells. Next, Haque et al. [46] studied and proved antibacterial activity of A. barbadensis gel ethanol extract against E. coli, Klebsiella pneumoniae, P. aeruginosa, and S. aureus. Ben Moussa et al. [47] investigated antimicrobial effectiveness of different extract of A. barbadensis gel against foodborne pathogens. Ethanol extract did exhibit best results as its inhibited growth of P. aeruginosa, S. aureus, and Aspergillus niger.
The objective of our research was to investigate the content of bioactive phytoconstituents (total phenols, proanthocyanidins, proteins), enzymatic (amylase, cellulase, lipase, peroxidase, protease, transglutaminase), and antioxidant activities of fresh and lyophilized A. arborescens and A. barbadensis and their ethanol extracts. Furthermore, research of antimicrobial effectiveness of lyophilized A. arborescens and A. barbadensis and their ethanol extracts was performed, since gel and juice of aforementioned aloe species already showed good inhibitory efficacy [1]. Growth inhibition of both Gram-positive and Gram-negative bacteria was demonstrated using ethanolic extracts of A. arborescens and A. barbadensis as inhibitors by the disk diffusion method alone. A study on comparison of unknown A. arborescens and well-known A. barbadensis has been done. This is the first study in which the activity of various enzymes has been determined, and it is important to emphasize the activity of cellulase and transglutaminase in all aloe samples. Moreover, it is the first study to quantify the antimicrobial activity of lyophilized aloes and their extracts. An important contribution to science is also the excellent inhibition of the growth of microorganisms with the addition of the less known lyophilized A. arborescens and its extract. The diversity of our samples and exceptional antimicrobial efficacy of these prove the versatility of using both aloes as antimicrobial agents.

Efficacy of Lyophilization and Extraction of A. arborescens and A. barbadensis
As can be seen from Table 1, 99.4% of water was removed from both types of Aloe spp. The lyophilization process was successful, as sources indicate that A. barbadensis contain approx. 99.3% of water [26]. Furthermore, extracts with bioactive substances from A. arborescens and A. barbadensis were obtained by Soxhlet extraction. Table 1 shows the extraction efficiency. The yield was slightly higher in the extraction of A. arborescens (0.233 g of extract/g of DW) than A. barbadensis (0.142 g of extract/g of DW).

Phenolics and Proanthocyanidins Content, Total Protein Concentration, and Antioxidant Activity of A. arborescens and A. barbadensis
The contents of bioactive compounds present in A. arborescens and A. barbadensis were determined by spectrophotometric methods. The results are presented in Table 2. The highest concentration of total phenols (TP) was detected in the ethanolic extract of A. barbadensis, while the extract of A. arborescens contained about five times lower TP content. The TP content in fresh and lyophilized samples was not detected as the concentrations were too low. Results are comparable to study from Vidic et al. [48], but they determined lower TP content in Soxhlet ethanol extract of Aloe spp. gel. The difference is probably due to the different preparation of the extracts, as we used lyophilized gel for extraction and determined higher TP content.
Like the TP content, the highest proanthocyanidin (PAC) content was detected in A. barbadensis extract. The PAC content is also about six times higher in A. barbadensis than in A. arborescens. Low concentrations of PAC were also detected in lyophilized samples, while they were not detected in fresh ones.
In terms of protein content, the highest concentration was present in the lyophilized samples, followed by ethanol extracts. Comparing the two aloes, A. arborescens showed highest total protein concentration in both cases.
Regarding antioxidant activity, fresh and lyophilized samples showed low percentage of inhibition, while ethanol extracts of A. arborescens and A. barbadensis showed a rapid decrease in absorbance and further highest percentage of inhibition were observed. The results for both aloes are similar and are completely comparable to already published studies [49]. Both ethanol extracts showed significant antioxidant activity.
The process of extracts preparation can also have a significant influence on the content of bioactive substances, as the degradation of thermally sensitive substances can also occur during Soxhlet extraction. Many factors, such as growing conditions, plant age, plant type, extraction processes, and analytical methods, can affect the presence of secondary metabolites in a plant and their biological activities [50]. Therefore, comparison of results from different studies is sometimes difficult or even impossible.

Enzymatic Activities of A. arborescens and A. barbadensis
Enzymes are highly specific biocatalysts involved in biotechnology. Plants are an important source of enzymes, especially those that are not naturally present in the human body (e.g., cellulase). Therefore, a study of various enzyme activities in different samples of A. arborescens and A. barbadensis was performed.
The activity of selected enzymes was determined in fresh and lyophilized gel of A. arborescens and A. barbadensis as well as in their ethanol extracts obtained by Soxhlet extraction. The stability of enzymes under extreme conditions (e.g., high temperature, pressure, solvent etc.) and the effect of medium factors are interesting for different industrial applications. Changes in protein structure may occur under extreme conditions. The spatial structure of many proteins may be significantly altered, causing denaturation and consequent loss in the activity. If conditions are less adverse protein structure may largely be retained. Minor structural changes may induce an alternative active protein state, which may possess altered activity, specificity, and stability [51,52]. The thermostability of enzymes is conditioned also by contributing factors, such as hydrogen bonds (intra-and intermolecular hydrogen bonds), electrostatic interactions, disulfide bonds, hydrophobic interactions, metal binding, deletion or shortening of loops, etc. However, there are no specific rules for thermostability of the enzymes. Among others, it may depend on the source from which the enzyme is derived [53]. The results of selected enzyme activities are presented in Table 3.
The highest α-amylase activity was present in the ethanol extract of A. arborescens and was almost four times higher than the activity in the ethanol extract of A. barbadensis. Comparing aloes, cellulase activity was higher in A. arborescens regardless of the type of the sample (fresh, lyophilized, extract), while lipase activity was only detected in lyophilized A. barbadensis and in its ethanol extract. Peroxidase activity was determined in all samples with higher value in A. barbadensis samples. The activity of the protease enzyme was determined in lyophilized A. arborescens and A. barbadensis and their extracts. The highest activity was achieved in the ethanol extract of A. barbadensis. The presence of the enzyme transglutaminase, which is particularly important in wound healing [54], was also detected in samples of both types of aloe. Slightly higher activity was present in A. arborescens samples. It should be emphasized that the difference in enzyme activities may appear due to the type of sample (fresh, lyophilized, extract), as the enzyme activity can be maintained or even increased by the lyophilization process [55]. The results of the study are remarkable, as plant materials such as aloe could be used to isolate enzymes for further applications. For example, α-amylase has gained much attention in recent years due to its ability to hydrolyze starch, which allows the inclusion of this enzyme in many applications, including in the baking industry, in environmentally friendly and safe detergents, and in the production of fructose syrup [56]. A particularly important contribution to the study is also the generally high cellulase activity in all samples, especially in the ethanolic extract of A. arborescens, as cellulase is an extremely applicative enzyme. The key areas of cellulase application in industry are currently beverages, detergents, food, healthcare, paper, and textiles. The potential of cellulases in the fight against antibiotic-resistant bacteria is also extremely interesting [57]. On the other hand, lipases isolated from plants represent potential for commercial applications in the food, detergent, and pharmaceutical industries, but their low expression in plants and difficulty in isolation limit their commercial applicability [58]. Furthermore, peroxidases are one of the key antioxidant enzymes used in the fields of environment, medicine, agriculture, and analytics. One of the more widespread applications is the use of horseradish peroxidase in the development of biosensors [59], while plant proteases are mostly used in bioactive peptide production, baking industry, dairy processing, and meat tenderization [60]. In addition to the previously mentioned important role of transglutaminase in wound healing, some research has also emerged on the potential use of plant transglutaminase as a food additive [61]. To the best of our knowledge, no similar comparative study in the literature that contains enzymatic activities in different samples of A. arborescens and A. barbadensis was found. Therefore, the obtained results present a major contribution to the identification of important enzymes from Aloe spp.

Qualitatively Determined Antimicrobial Activity of A. arborescens and A. barbadensis
Using the disk diffusion method on nutrient agars, the inhibitory property of lyophilized aloes and their ethanol extracts was qualitatively determined. Tested pathogenic microorganisms cause various infections, e.g., Candida fungus is one of the most common causes of fungal infections (candidiasis) and in addition, it is one of the most often tested on cosmetic products, besides E. coli, S. aureus, and P. aeruginosa [62,63].
The disk diffusion method showed the antimicrobial efficacy of A. arborescens and A. barbadensis only in the case of ethanol extracts. Lyophilized samples did not indicate inhibitory properties as no inhibition zone was detected.
Ethanol extracts of both aloes showed inhibitory properties at Gram-positive bacteria B. cereus and Gram-negative bacteria E. coli and P. fluorescens, while the growth of other tested microbial (C. albicans, P. aeruginosa, S. aureus) cultures was not inhibited. Table 4 shows the microbial growth inhibition zone in the case of using ethanol extracts of A. arborescens and A. barbadensis as inhibitors. From Table 4, it can be seen that the growth of E. coli was better inhibited by the ethanol extract of A. barbadensis than in the case of A. arborescens at both initial concentrations (10 6 and 10 7 CFU/mL). Comparing our results to other studies, their ethanol extract of A. barbadensis did not show any inhibition [16,20], or the growth inhibition was much lower [34]. Growth inhibition of P. fluorescens was perceived in other studies only in the case of A. barbadensis methanol extract which contained aloe emodin and has already been shown to have antimicrobial effect [3]. In our study, both ethanol extracts were confirmed to be good antimicrobial agents. The growth of P. fluorescens was slightly better inhibited by the ethanol extract of A. arborescens with somewhat larger inhibition zone. At higher initial concentration (10 7 CFU/mL) of P. fluorescens, the inhibition zone was not observed. In the case of B. cereus growth, the inhibition zone was equal for both aloes regardless of the initial concentration, meaning that they are equally effective antimicrobial agents for this microorganism. The results are comparable to the study, where an inhibition zone larger than 8 mm at higher initial concentration of microorganism was determined [34].

Quantitatively Determined Antimicrobial Activity of A. arborescens and A. barbadensis
Since there is a lack in studies that would contain quantitative antimicrobial activity, A. arborescens and A. barbadensis were further tested by the broth microdilution method. This offers quantitative results to determine the microbial growth inhibition rate (MGIR) at different concentrations of the added antimicrobial sample. In our study, samples used as antimicrobial agents were ethanol extracts from A. arborescens and A. barbadensis, as well as lyophilized aloes. The inhibitory efficacies of A. arborescens and A. barbadensis on the growth of microbial cells were tested at a specific initial concentration (see Figure 2 caption for details) of each microorganism.

Lyophilized A. arborescens and A. barbadensis
By lyophilization, the moisture content of A. arborescens and A. barbadensis was reduced to a minimum (99.4% of water was removed) while no thermosensitive substances were destroyed, and most importantly, biological activity of the compounds present in lyophilized samples was mantained. Due to the lyophilization process, the sample is more stable because no water is present. These advantages are also exploited in the storage of samples. Very few studies [16,35,64] on the antimicrobial activity of lyophilized A. arborescens and A. barbadensis were described in the reviewed literature, and thus, the study of the antimicrobial efficacy of lyophilized aloes was performed in our research. Figure 2 shows MGIR for B. cereus, C. albicans, E. coli, P. aeruginosa, P. fluorescens, and S. aureus in the case of using lyophilized A. arborescens and A. barbadensis as inhibitors.  (f) MGIRs for S. aureus; Initial concentrations of microbial cultures: B. cereus 10 7 CFU/mL, C. albicans 10 6 CFU/mL, E. coli 10 7 CFU/mL, P. aeruginosa 10 7 CFU/mL, P. fluorescens 10 7 CFU/mL, and S. aureus 10 5 CFU/mL. The numbers in boxes indicate the highest MGIR for specific sample. Data expressed as mean ± standard deviation of three replicates that vary for max. 3%.
Both lyophilized A. arborescens and lyophilized A. barbadensis (600 µg sample/mL suspension) inhibited the growth of P. aeruginosa most effectively. The lowest MGIR for lyophilized A. arborescens sample was shown in the case of E. coli (61 ± 1% MGIR) and the lyophilized A. barbadensis sample by C. albicans (46 ± 1% MGIR). The growth of B. cereus was better inhibited by lyophilized A. barbadensis (90 ± 2% MGIR) than by A. arborescens (77 ± 1% MGIR). Quite a difference was shown in the inhibition of the growth of the yeast C. albicans, as the lyophilized A. arborescens inhibited its growth with 74 ± 2% MGIR while the lyophilized A. barbadensis with only 46 ± 1% MGIR. Regarding growth inhibition of E. coli, lyophilized A. barbadensis exhibited 51 ± 3% MGIR, which is lower than beforementioned MGIR in the case of A. arborescens. The growth inhibition of P. aeruginosa is particularly prominent, as the samples almost completely (99 ± 1% MGIR at A. arborescens and 98 ± 1% MGIR at A. barbadensis) inhibited the growth of their strains. The growth of P. fluorescens was equally inhibited by lyophilized A. arborescens and A. barbadensis. In a recent study, Habeeb and others [33] determined a minimum inhibitory concentration (MIC) of lyophilized A. barbadensis 25000 µg/mL at the initial concentration 10 5 CFU/mL of the S. aureus microorganism. In our study, at the same initial concentration of S. aureus, a 60 ± 1% MGIR was achieved, with the addition of 600 µg/mL lyophilized A. barbadensis, which proves that a lower concentration of lyophilized A. barbadensis can already inhibit the growth of S. aureus. A. arborescens inhibited the growth of S. aureus strains even more effectively (79 ± 2% MGIR).
Regarding the inhibition of microbial cells at lower concentrations of added inhibitory agent, the effect of A. arborescens and A. barbadensis on the growth of P. aeruginosa should be emphasized. The addition of 500 µg/mL of lyophilized aloes achieved above 90% MGIR. Moreover, the growth of P. aeruginosa was impaired with the addition of even lower concentrations.
Both lyophilized aloes seem to be good antimicrobial agents. In addition to the determination of MIC values for S aureus [33], other MIC determinations for lyophilized aloes were not found in the reviewed literature. Based on the obtained results, the MIC 90 value for lyophilized A. barbadensis in the case of B. cereus was 600 µg/mL, while both lyophilized samples showed exceptional antibacterial efficacy on P. aeruginosa. The MIC 90 value for A. arborescens was 457 µg/mL and for A. barbadensis 395 µg/mL. For other microbial cells, further studies with added higher sample concentrations would be required to determine the MIC values. In general, A. arborescens showed in common better microbial growth inhibition because of a higher MGIR for all microbial cultures except B. cereus.
Quantification of the MGIR at four different concentrations of lyophilized samples of A. arborescens and A. barbadensis against fungi, Gram-positive and Gram-negative bacteria, has not been performed in any study to date.

Ethanol Extracts of A. arborescens and A. barbadensis
Most published research on the topic of antimicrobial activity of aloes involves testing different extracts as inhibitors. In our study, we used ethanol as a solvent in the extraction process as it is known to be effective, efficient, and safe. Figure 2 shows MGIR in the case of using ethanol extracts of A. arborescens and A. barbadensis as inhibitors. Ethanol extracts of A. arborescens and A. barbadensis (600 µg of sample/mL of suspension) showed an extremely high MGIR in the case of all tested microbial cells. The highest inhibition of A. arborescens ethanol extract was detected for the growth of P. aeruginosa (96 ± 3% MGIR) and the lowest for the growth of C. albicans (47 ± 2% MGIR), respectively. Ethanol extract of A. arborescens also inhibited growth of E. coli with 79 ± 2%, P. fluorescens with 80 ± 3%, B. cereus with 83 ± 2%, and S. aureus with 93 ± 1% MGIR. Ethanol extract of A. barbadensis gave the highest inhibition for growth of B. cereus (99 ± 1% MGIR) following for the growth of P. aeruginosa (98 ± 2% MGIR). Moreover, A. barbadensis extract inhibited S. aureus and P. fluorescens with 95 ± 1% MGIR. Growth of E. coli was inhibited with 85 ± 2% MGIR and growth of C. albicans with 30 ± 3% MGIR.
In the case of ethanol extract as an inhibitor, A. barbadensis extract was generally slightly better than A. arborescens as it achieved higher inhibition rate for almost all tested microorganisms (except C. albicans, where the MGIR was only 30 ± 3%).
Therefore, it is interesting to compare the results at a lower added concentration of A. arborescens and A. barbadensis ethanol extracts. Since MGIRs with the addition of 500 µg/mL ethanol extract were still high especially for B. cereus, P. aeruginosa, P. fluorescens, and S. aureus, it is important to consider the results of the MGIR with the addition of 80 and 200 µg sample/mL suspension.
The results of our antimicrobial efficacy study of A. arborescens and A. barbadensis ethanol extracts show that both samples at least slightly inhibited the growth of all microbial cells, with the addition of 200 µg/mL inhibitory agent. Ethanol extract of A. arborescens with the concentration of 80 µg/mL did not inhibit the growth of E. coli and C. albicans, and ethanol extract of A. barbadensis did not inhibit the growth of C. albicans strains. In both cases, ethanol extracts proved to be the strongest antimicrobial agents among all tested samples in inhibiting the growth of S. aureus, as they achieved 72 ± 3% and 73 ± 1% MGIR at a concentration of 80 µg/mL, respectively.
Already published studies provide different MIC values when using ethanol extracts of A. barbadensis as antimicrobial agents. MIC values for E. coli are in the range of 10000-125 µg/mL [22,38,46], for S. aureus between 500-125 µg/mL [22,38,46], for P. aeruginosa between 650-100 µg/mL [22,41,46], and for C. albicans strains about 400 µg/mL [38]. MIC values may differ between studies mainly due to different ethanol extract preparation procedures. In most cases, however, the initial concentrations of microbial cultures are unknown. The MIC values for B. cereus and P. fluorescens were not found in the literature reviewed, while MIC 90 values for the mentioned microorganisms were successfully determined in our study.
The MIC 90 value for the ethanol extract of A. barbadensis in the case of B. cereus is 432 µg/mL and in the case of P. fluorescens is 538 µg/mL. Higher concentrations should be tested to determine the MIC value for ethanol extract of A. arborescens. A MIC 90 value was also determined for both extracts in the case of P. aeruginosa (493 µg/mL for A. arborescens and 558 µg/mL for A. barbadensis) and S. aureus (575 µg/mL for A. arborescens and 562 µg/mL for A. barbadensis).
While some antimicrobial studies of A. barbadensis extracts can be found in the literature, the quantification of antimicrobial efficacy of A. arborescens extract is a major contribution to this research field. In general, the ethanol extract of A. barbadensis had a slightly more effective growth inhibition against all microorganisms except on yeast C. albicans, where the higher MGIR has been reached with the ethanol extract of A. arborescens.

Discussion
A comparative study of different samples of A. arborescens and A. barbadensis was performed.
Numbers of various secondary metabolites are found in plants which contribute to compelling biological activities. Our study shows the presence of different important phytoconstituents in A. arborescens and A. barbadensis. Ethanol extract for which the contents of total phenols and proanthocyanidins were determined also showed good antioxidant activity. In various studies [65][66][67][68], a number of potential antioxidant and antimicrobial components have been isolated from Aloe species, which most likely contribute to the biological activity of Aloe with a synergistic effect. Additional studies with LC-MS have also been previously performed, where mostly anthraquinones, phytosterols, alkaloids, and fatty acids were identified [69,70].
The presence of enzymes in A. arborescens and A. barbadensis was also demonstrated. Extracts and lyophilized samples in particular showed higher enzyme activities. The presence of the enzyme transglutaminase, which is involved in stabilization, general physiology, and repair of many areas of tissue (e.g., skin) [54], should be emphasized. The presence of transglutaminase in all samples proves that aloe can contribute to better and faster wound healing [71].
Using a qualitative disk diffusion method, the antibacterial efficacy on the growth of both Gram-positive and Gram-negative bacteria of ethanol extracts was determined. Further, antimicrobial activity of A. arborescens and A. barbadensis was determined quantitatively. Interestingly all of A. arborescens samples showed the highest inhibition for the growth of P. aeruginosa, as did the lyophilized A. barbadensis sample. Furthermore, ethanol extract of A. barbadensis gave the highest inhibition for the growth of B. cereus.
Previously, various preparations of A. vera including creams, juices, and gels have been used as a traditional medicine in some parts of the world to treat various diseases [72]. The fact that A. arborescens and A. barbadensis are effective antimicrobial agents was also reinforced by performed study. A. arborescens and A. barbadensis are possible solution as antimicrobials for different applications in food production. For example, their extracts or potentially isolated antimicrobial compounds can be incorporated in different package materials to prevent or inhibit microbial growth and, moreover, can be extensively used in cosmetic and pharmaceutical products, as in natural food and dietary supplements. Additionally, the combination of antimicrobial efficacy and the presence of transglutaminase in the obtained samples show the even greater potential of aloe preparations in medical application.
E. coli, found in the human gastrointestinal tract, lives in a mutually beneficial relationship with the host, but it is also one of the most common pathogens in humans as it is responsible for a wide range of diseases [73,74]. It is a food-borne pathogen, and with the extensive use of antibiotics, food-borne pathogens develop antibiotic resistance which means many food-borne illnesses and thus a lack of effective treatment [75]. The best inhibitory properties for E. coli growth have been found to have A. barbadensis ethanol extract and right after there is A. arborescens ethanol extract. Comparing results with our published study (with fresh gels and juices from aloes) [1], better antimicrobial efficacy on E. coli growth displayed all A. barbadensis samples (except lyophilized).
Although fresh juice of A. barbadensis and A. arborescens does not inhibit the growth of S. aureus [1], an excellent inhibition for the same microbial species was found for ethanol extracts of both aloes. However, a better growth inhibitor of most selected microorganisms was the extract of A. barbadensis; lyophilized A. arborescens, however, offered better inhibition of S. aureus growth than lyophilized A. barbadensis. Multidrug resistance is a wellknown problem in medicine, and S. aureus is perhaps the bacterium of the highest concern due to its virulence and ability to cause a diverse set of life-threatening infections [76,77]. Our research has shown that A. arborescens and A. barbadensis have a great potential in terms of reducing possible infections with S. aureus strains.
In addition, research showed all samples to be good growth inhibitors of B. cereus, a pathogenic spore-forming bacterium that is often associated with food-borne diseases as spores can also survive pasteurization and cooking and multiply when foods are stored incorrectly [78,79]. Best inhibitory properties for the growth of B. cereus showed A. barbadensis ethanol extract. Comparing the results of both aloes for inhibition of this microorganism, MGIRs of A. barbadensis were higher for all samples.
The tested samples from this study and our previous one [1] were found to be good inhibitors of P. fluorescens growth, which is opportunistically pathogenic and able to reside in many environments [80]. The best inhibitory growth properties for this microorganism showed A. barbadensis ethanol extract. A comparison of the inhibitory effect of both aloes shows that A. barbadensis samples have a better antimicrobial effect on P. fluorescens growth than A. arborescens samples. Lyophilized samples and fresh gels of both aloes have a similar inhibitory effect on growth of P. fluorescens. In common, all tested inhibitors showed satisfactory results for the P. fluorescens growth inhibition study.
Further, the best inhibitory properties for the growth of the yeast C. albicans have fresh A. barbadensis gel [1] and lyophilized A. arborescens. Comparing the results of both aloes, A. barbadensis showed to have a better antimicrobial effect on the growth of C. albicans, which is the fourth leading cause of bloodstream infections [81].
According to obtained results, all samples were also good inhibitors of P. aeruginosa growth. Therefore, A. arborescens and A. barbadensis could reduce the possible infections with P. aeruginosa, which has a leading role among infections caused by Gramnegative strains [82]. Due to its nutritional versatility, high number of virulence factors, and high antibiotic resistance, treatment is extremely difficult [83]. Both, A. barbadensis and A. arborescens gave high inhibition level for the growth of P. aeruginosa irrespective of the form of the sample (fresh, lyophilized, or ethanol extract) [1]. These results are very important as P. aeruginosa is one of the first three causes of opportunistic infections in humans [84].
For the first time, the enzymatic and antimicrobial activity of lyophilized A. arborescens and A. barbadensis and their extracts were quantitatively determined. A comprehensive study confirmed the presence of versatile enzymes in A. arborescens and A. barbadensis as well as the growth inhibition rates for six microorganisms, representatives of fungi, Gramnegative and Gram-positive bacteria, with the addition of four different concentrations of A. arborescens and A. barbadensis, as inhibitors. The results confirm and give credence to the beneficial effects of using A. arborescens and A. barbadensis and their extracts.

Plant Material and Preparation of Samples
The gels of A. arborescens and A. barbadensis were obtained from fresh mature leaves of A. arborescens and A. barbadensis. For one batch, 5 leaves (345.2 g of A. arborescens and 390.7 g of A. barbadensis) of each aloe, long between 50-60 cm, were washed under running water and thick outer layers of the leaves were separated using a knife. 165.3 g of A. arborescens and 214.1 g of A. barbadensis inner cores were cut into approx. 1 cm 3 big pieces and centrifuged (Eppendorf ® Centrifuge 5840R, Wesseling, Deutschland) at 11,000 rpm, room temperature for 15 minutes. The supernatant (juice) was removed and 149.8 g of A. arborescens and 197.5 g of A. barbadensis fresh transparent gel was collected and homogenized (Tehtnica ® Rotamix 701 MD, Železniki, Slovenia). For one batch, 148.5 g of fresh A. arborescens gel and 196.6 g of fresh A. barbadensis gel were subjected to a lyophilization process Kambič ® Freeze Dryer LIO 2000 PNS, Semič, Slovenia) to remove water. Thus, lyophilized A. arborescens (0.9 g DW) and A. barbadensis (1.2 g DW) were obtained. As the samples of lyophilized aloes were too dry to apply, they were diluted with a minimal amount of distilled water before use and homogenized. Lyophilized A. arborescens and A. barbadensis were always prepared at a concentration of 0.1 g/mL.
Furthermore, using the Soxhlet apparatus, extractions of lyophilized gel of A. arborescens and A. barbadensis were performed. For one batch, the lyophilized gel of A. arborescens (0.9 g DW) or A. barbadensis (1.2 g DW) was placed in a porous bag made from a strong filter paper, which was placed in Soxhlet extractor. A volume of 150 mL of ethanol was used as an extraction solvent. The solvent was further evaporated using rotavapor (Büchi ® Rotavapor R-144, Flawil, Switzerland) and ethanol extracts of A. arborescens (0.21 g) and A. barbadensis (0.17 g) were obtained. Extracts were stored at 4 • C until use. Ethanol extracts of A. arborescens and A. barbadensis were always prepared at a concentration of 0.1 g/mL in 5% DMSO.

Determination of Total Phenolics (TP) Content
The content of TP was determined using Folin-Ciocalteu's reagent. A volume of 0.5 mL of the prepared sample solution (2 g/L) was mixed with 2.5 mL of FC solution, previously diluted with distilled water in 1:10 ratio. Further, 2 mL of Na 2 CO 3 solution with concentration of 75 g/L was added to each sample; samples were then incubated for 5 min in a water bath at 50 • C. The solutions were cooled to room temperature, and the absorbance was measured at 760 nm. Following a similar procedure, a standard curve with gallic acid was prepared. The results are expressed as mg of GA per g of sample. The experiments were performed in triplicates, and the results represent the mean values and standard deviations.

Determination of Proanthocyanidins (PAC) Content
The PAC content was determined by the calorimetric method using hydrochloric acid and n-butanol. To 1 mL of the prepared sample solutions (5 mg/mL), 10 mL of FeSO 4 × 7 H 2 O in a mixture of HCl and n-butanol (2:3) was added. The prepared solutions were incubated for 15 min in a water bath at 95 • C. The absorbance of the cooled samples was measured at 540 nm. Based on the measured absorbance, the mass concentration of PAC was calculated, expressed as mg of PAC per g of sample. All experiments were performed in triplicates, and the results represent the mean values and standard deviations.

Determination of Antioxidant Activity
Antioxidant activity was determined using DPPH method [65]. A 77 µL of prepared sample solutions (1 mg/mL) and 3 mL of a DPPH solution prepared in methanol (6 ×10 −5 M) were mixed. The solutions were then incubated for 15 min at room temperature in the dark, and the absorbance at 515 nm was immediately measured. Antioxidant activity is expressed as a percentage of inhibition relative to the reference solution, containing 77 µL of methanol and 3 mL of prepared DPPH solution [85]. All experiments were performed in triplicates, and the results represent the mean values and standard deviations.

Determination of Total Protein Concentration
The total protein concentration in A. arborescens and A. barbadensis samples was determined by Bradford method [86] using bovine serum albumin as a standard. A volume of 1 mL of Bradford reagent was pipetted into a microcentrifuge, and 20 µL of sample was added. The solutions were stirred immediately and incubated for 15 min at room temperature; then, their absorbance at 595 nm was measured. Following a similar procedure, a standard curve with BSA was prepared. The results are expressed as mg of proteins per g of sample. All experiments were performed in triplicates, and the results represent the mean values and standard deviations.

Determination of Enzyme Activities
Activities of selected enzymes were defined using specific spectrometric activity assays. All experiments were performed in triplicates.
• α-Amylase activity was determined by the DNS method [87] with starch as the substrate and maltose as the standard. A volume of 0.5 mL of prepared sample solutions and 0.5 mL of 1% (w/v) starch solution prepared in sodium buffer solution was pipetted into suitable centrifuge tubes. Mixture was incubated for 3 min at 20 • C; then, color reagent (prepared with 5.3 M potassium sodium tartrate, tetrahydrate, and 96 mM DNS solution) was added. Covered containers were incubated in a boiling water for 15 min. A volume of 10 mL of distilled water was added to cooled solutions and mixed by inversion. The absorbance was measured at 540 nm. Results are expressed as units per gram of sample; one unit will liberate 1 mg of maltose from starch in 3 min at 20 • C at pH 6.9. • Cellulase activity was measured using glucose as substrate [88]. A volume of 4 mL of a Sigmacell solution was pipetted into suitable containers, then 1 mL of sample was added. Mixture was incubated for 120 min at 37 • C with moderate shaking. Further, suspension was transferred into iced water bath. When suspension was settled, it was centrifuged at 11,000 rpm for 2 min, and 100 µL of supernatant was added to 3 mL of glucose solution. Absorbance was measured at 340 nm for 5 min, and the increase in absorbance was used to determine enzyme activity. Results are expressed as units per gram of sample; one unit liberates 1 µmol of glucose from cellulose in one hour at 37 • C and pH 5.0. • Lipase activity was determined using p-NPB as substrate [89]. A volume of 0.9 mL of 100 mM sodium phosphate buffer with 150 mM sodium chloride and 0.5% triton was pipetted into suitable containers. A volume of 0.1 mL of sample was added, and the mixture was incubated for 5 min at 37 • C. Further, 0.01 mL of 50 mM p-NPB was added, and absorbance was measured at 400 nm for 5 min. The increase in absorbance was used to determine enzyme activity. The results are expressed as units per gram of sample; one unit will release 1 nmol of p-nitrophenol per minute at 37 • C and pH 7.2 using p-NPB.

•
Peroxidase activity was determined using H 2 O 2 as an inhibitor [90].

Disc Diffusion Method
The disk diffusion method [93] was used to qualitatively determine the susceptibility of microorganisms to different antimicrobial samples. The antimicrobial efficacy of A. arborescens and A. barbadensis on bacteria and fungi were performed at optimal conditions and two initial concentrations of each microorganism as in our previous study [1]. The indicator of the inhibitory property of the sample was the inhibition zone shown at the sample. The diameter of the growth inhibition zone was measured to compare the antimicrobial efficacy of the two aloes on the growth of different microbial cultures. dH 2 O and 5% DMSO were used as negative controls. Vancomycin and amoxicillin (30 µg/disc) were used as positive controls. All experiments were performed in triplicates.

Broth Microdilution Method
A broth microdilution method [94] was used to quantitatively analyze and thereby determine the microbial growth inhibition rate (MGIR) at different sample concentrations (80,200, 500, and 600 µg of sample/mL of microbial suspension). MGIRs were determined based on optical density of the growth control and sample to determine the percentage of microbial growth inhibition [1]. Additionally, MIC 90 values were determined experimentally or calculated, as concentrations where the samples inhibited the growth of microbes by 90% MGIR [1]. All experiments were performed in triplicates.

Conclusions
The results of our research demonstrated the presence of bioactive substances such as phenolics, proanthocyanidins, and enzymes in samples of A. arborescens and A. barbadensis. Particularly more concentrated samples, such as extracts or lyophilized ones, are rich in biologically active ingredients and exhibit high antioxidant potential. Furthermore, the results have proved A. arborescens and A. barbadensis to hold excellent potential as antimicrobial agents. Both aloes inhibited the growth of B. cereus, C. albicans, E. coli, P. aeruginosa, P. fluorescens, and S. aureus, representatives of Gram-positive, Gram-negative bacteria, and fungi. Further, for most tested microorganisms (B. cereus, E. coli, P. fluorescens, S. aureus) the best inhibitory effect was found for ethanol extracts of A. barbadensis and A. arborescens. The best inhibitory properties of the tested samples (even the fresh ones) were shown for P. aeruginosa growth. P. aeruginosa can cause a wide range of infections (respiratory tract, urinary tract, skin infections, superficial structures of the eye, etc.), prevailing infections of wounds and burns. According to the obtained results, the use of personal hygiene products such as soaps and various creams containing A. arborescens and A. barbadensis could reduce the possible infections with P. aeruginosa and other tested microorganisms.
As both aloes are rich in essential and bioactive nutrients as phytochemicals, enzymes, and other compounds, they need to be utilized as much as possible. Functional foods and food supplements from natural sources like A. arborescens and A. barbadensis, are a good possibility to intake essential and bioactive molecules and nutrients in the human body since they are natural and not synthesized and therefore more receptive to the human body. Further, incorporation of both A. arborescens and A. barbadensis into different materials as antimicrobials could be used for applications in drug delivery, wound healing, etc. Such materials have a high potential to reduce microbial growth, and they could make a huge contribution to the food, medicinal, and pharmaceutical industries. Furthermore, characterization and isolation of individual bioactive constituents from A. vera extracts should be performed to determine the antimicrobial efficacy of the individual components. In addition, toxicity studies of A. vera extracts should be performed to determine the safety indices of the extracts. Clinical trials should also be conducted to investigate the potential of A. vera extracts in the treatment of, e.g., bacterial diseases.
No. 2187/FS-2019 and by Ministry of Education, Science and Sport, The Public Scholarship, Development, Disability and Maintenance Fund of the Republic of Slovenia within the frame of Students Innovative Projects for the Benefit of Society contract number: 11083-25/2017 (Active ingredients from Aloe vera, their use and impact on health in the third period of life).

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available in article.

Conflicts of Interest:
The authors declare no conflict of interest.